Methods And Compositions For Detection Of Cowden Syndrome (CS) And CS-Like Syndrome

ABSTRACT

In one aspect, the invention is directed to a method of detecting Cowden syndrome (CS) or CS-like syndrome in an individual comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is positive for CS or CS-like syndrome. In another aspect, the invention is directed to a method of determining whether an individual is at risk for developing Cowden syndrome (CS) or CS-like syndrome comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is at risk for developing for CS or CS-like syndrome. In yet another aspect, the invention is directed to an article of manufacture for detecting Cowden syndrome (CS) or Cowden-like syndrome in an individual, comprising one or more agents that detects mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, and instructions for use.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 14/947,851 filed on November 20, 2015, which is a continuation of U.S. application Ser. No. 13/445,688 filed on Apr. 12, 2012 which is a continuation of U.S. application Ser. No. 12/462,053 filed on Jul. 27, 2009, which claims the benefit of U.S. Provisional Application No. 61/137,042, filed on Jul. 25, 2008. The entire teachings of the above applications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant 1PO1CA124570-01A1 from the National Cancer Institute. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listing contained in the following ASCII text file:

a) File name: 37861044004SEQLIST.txt; created Oct. 3, 2017, 1.46 KB in size.

BACKGROUND OF THE INVENTION

Phosphatase and tensin homolog deleted on chromosome ten (PTEN [MIM 601728]) is a ubiquitous tumor suppressor that plays a role in both heritable and sporadic neoplasias (Zbuk, K. M., et al. Nat. Rev. Cancer, 7:35-45 (2007)). Cowden syndrome (CS [MIM 158350]) is a difficult to recognize, autosomal dominant inherited cancer syndrome characterized by benign and malignant breast, thyroid and endometrial neoplasias in addition to cutaneous findings and macrocephaly (Eng, C., Hum. Mut., 22:183-198 (2003)). Germline PTEN mutations have been found in 85% of those with classic CS while 15% remain mutation negative despite extensive analyses including the promoter and looking for large deletions and rearrangements (Marsh, D. J., et al., Hum. Mol. Genet., 7:507-515 (1998); Zhou, X. P., et al., Am. J. Hum. Genet., 73:404-411 (2003)). Many more patients with features reminiscent of CS, not meeting diagnostic criteria (National Comprehensive Cancer Center Practice Guidelines (NCC); Table 1) and referred to as CS-like, are evaluated by clinicians for CS and cancer-risk. CS is believed to be without genetic heterogeneity (Nelen, M. R., et al., Nat. Genet., 13:114-116 (1996)) to date, only PTEN has been implicated in this syndrome. However, there likely exist other susceptibility genes for CS and CS-like phenotypes, especially in the latter, which appear to be a heterogeneous disease. Therefore, a better understanding of CS syndrome and CS-like syndrome is needed in order to provide better detection methods for these syndromes.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a method of detecting Cowden syndrome (CS) or CS-like syndrome in an individual comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is positive for CS or CS-like syndrome.

In another aspect, the invention is directed to a method of determining whether an individual is at risk for developing Cowden syndrome (CS) or CS-like syndrome comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is at risk for developing for CS or CS-like syndrome.

In yet another aspect, the invention is directed to an article of manufacture for detecting Cowden syndrome (CS) or Cowden-like syndrome in an individual, comprising one or more agents that detects mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the experimental design for SDH mutation testing and functional analysis. Note PTEN gene test encompasses intragenic PCR-based mutation analysis, promoter and large deletion analysis. From the 2,270 PTEN mutation negative CS/CS-like individuals, the most proximal (i.e., most recent) consecutive 375 PTEN mutation negative subjects were selected to proceed to MnSOD expression analysis. It is these 375 subjects that represent the series for this SDH study.

FIGS. 2A-2E show genetic and biochemical analyses of CS/CS-like patients without germline PTEN mutations reveal a subset with germline SDH mutations resulting in biochemical dysfunction. FIG. 2A shows dot blots to screen for increased MnSOD protein levels. Boxed dots represent controls with low MnSOD levels. FIG. 2B shows illustrative sequencing chromatograms of germline heterozygous mutations of SDH genes identified in patients with CS/CS-like phenotypes (mutations as noted above each chromatogram). The germline mutations/variants are heterozygous manifested by overlapping peaks (arrows). FIG. 2C shows increased ROS in peripheral lymphoblasts from an individual with germline SDHD His5OArg. Increased. ROS levels are measured by increased carboxy-H2DCFDA staining as seen in cultured lymphoblast cells from the patient with SDHD His50Arg mutation denoting 1.5-fold higher ROS levels (middle) compared to a lymphoblast cell line derived from a normal control individual (left; P<0.001, Student T-test, 3 replicates). Finally, a control lymphoblast cell line treated with tert-butyl hydroperoxide for 90 min was used as a super-positive control and supra-induced ROS expression is noted by markedly increased carboxy-H2DCFDA staining (right). FIG. 2D shows protein expression of P-AKT and P-MAPK (P-ERK44/42) in germline heterozygous PTEN mutation positive individuals. Note different mutations result in varying activation of P-Akt and/or P-MAPK. FIG. 2E shows germline protein expression of PTEN, actin (loading control), P-Akt and P-MAPK (as labeled, from top to bottom). Fold change values beneath the P-Akt and P-MAPK blots represent the mean of normalized densitometrically obtained expressional levels of patient sample(s) relative to controls. In other words, (Patient P-Akt or P-MAPK intensity/corresponding patient actin intensity)/(Control P-Akt or P-MAPK intensity/corresponding control actin intensity). The ratio of control P-Akt or P-MAPK intensity to control actin intensity was normalized to 1.0. This type of quantitation (taking the ratio of the ratios) was chosen because it results in the most conservative (i.e., under-estimated) fold changes.

FIG. 3 shows a proposed Model for the Final Common Pathway of Putative Mitochondrial Dysfunction Resulting from Either PTEN or SDHx Mutation in Cowden and Cowden-like Syndromes. A simplified version of the signaling pathways involved in tumorigenesis in the setting of dysfunctional PTEN or SDH (represented by hatched colors). These pathways cross-talk leading to the final common outcome of tumor angiogenesis, cell proliferation. and inhibition of apoptosis. Note that one of the functions of SDH is the conversion of succinate to fumarate as part of the Kreb's tricarboxylic acid cycle. SDH dysfunction will therefore lead to an accumulation of succinate, which inhibits prolyl hydroxylases (PHD) and subsequently leads to the stabilization of HIF-1a. The stabilization of the latter also occurs a number of steps downstream of dysfunctional PTEN signaling. It is interesting to note that activated Akt (P-Akt) can increase ATP levels which result in increased ROS, presumably via mitochondrial dysfunctional signaling. This is postulated to set up a double feedback loop linking both the PTEN and SDH pathways.

FIG. 4 shows a suggested algorithm for clinical PTEN and SDH testing for CS/CS-like individuals. Also, see text for details.

DETAILED DESCRIPTION OF THE INVENTION

Clues to disease etiology are often obtained by examining whether certain CS/CS-like clinical features resemble those in other syndromes, by examining downstream signaling, and/or by looking at phenotype in murine models. In this situation, the murine model only vaguely resembles human CS (Zbuk, K. M., et al. Nat. Rev. Cancer, 7:35-45 (2007)). One prominent feature in the mouse model is pheochromocytoma, a neoplasia of the adrenal medulla, and its closely related neural crest-derived paraganglioma (PGL) (Satmbolic, V., et al., Cancer Res., 60:3605-3611 (2000). Pheochromocytoma and PGL are not known component features of CS (NCCN; Table 1).

Succinate debydrogenase (SDH) belongs to mitochondrial complex II, which participates in both the electron transport chain and the Kreb's cycle (reviewed by Eng, C., et al., Nat. Rev. Cancer, 3:193-202 (2003)). SDH comprises four subunits, SDHA, B, C and D, each of which is encoded by autosomal genes on 3 different chromosomes. While homozygous/compound heterozygous mutations in SDHA (MIN 600857) cause severe neurological dysfunction and cardiomyopathy, heterozygous germline mutations in SDHB-D (MIM 185470, 602413, 602690) cause a pheochromocytoma-PGL syndrome (Neumann, H. P. H., et al., N. Engl. J. med., 346:1459-1466 (2002)). Approximately 1%-5% of carriers of SDHB or SDHD mutations have been found to have renal cell carcinoma or papillary thyroid cancer (Vanharanta, S., et al., Am. J. Hum. Genet., 74:153-159 (2004); Benn, D. E., et al., J. Clin. Endocrinol. Metab., 91:827-836 (2006)) which are also features of CS. Fumarate hydratase (FH) is the enzyme immediately downstream of SDH. Homozygous germline mutations cause severe neurological dysfunction and death while heterozygous mutations are associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) (Launonen, V., et al., Proc. natl. Acad. Sci., USA, 98:3387-3392 (2001)). In vitro evidence also suggests that mitochondrial caspases and HIF1 are downstream molecules of the PTEN pathway (Zundel, W., et al., Genes Develop., 14:391-396 (2000); Tang, Y., et al., Cancer Res., 66:736-742 (2006)). The invention described herein is based, in part, on the discovery that SDHx represent susceptibility genes, other than PTEN, for CS/CS-like syndromes.

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer-risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. As described herein, SDHB-D are susceptibility genes for some PTEN mutation negative individuals with CS-like cancers.

Specifically, germline SDHB-D mutation analysis in 375 PTEN mutation negative CS/CS-like individuals was performed followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Amongst these, 10 (13.5%) had germline mutations/variants in SDHB (N=3) or SDHD (7), not found in 700 controls (P<0.001). Compared to PTEN mutation positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH vs, 30/107 PTEN, p<0.001), thyroid (5/10 vs. 15/106, <0.001) and kidney (2/10 vs. 4/230, p=0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants showed increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid and renal cancers beyond those conferred by germline PTEN mutations. Thus, SDH testing can be used for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid and/or renal cancers.

Accordingly, in one aspect, the invention is directed to a method of detecting (diagnosing) Cowden syndrome (CS) or CS-like syndrome in an individual comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is positive for CS or CS-like syndrome (indicates a diagnosis of CS or CS-like syndrome in the individual).

In another aspect, the invention is directed to a method of determining whether an individual is at risk for developing Cowden syndrome (CS) or CS-like syndrome comprising detecting the presence of a mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, wherein detection of a mutated SDHB, SDHD or a combination thereof indicates that the individual is at risk for developing for CS or CS-like syndrome.

As used herein, a “mutated SDHB” or a “mutated SDHD” is a SDHB or SDHD that has a sequence (e.g., nucleic acid (e.g., DNA, such as genomic DNA; RNA) sequence, amino acid sequence) that differs from, or is a variant of, the normal or wild type SDHB sequence or SDHD sequence. In one aspect of the invention, the mutated SDHB has a nucleic acid sequence that encodes an amino acid sequence comprising an Ala3Gly mutation, a Ser163Pro mutation or a combination thereof. In another aspect of the invention, the mutated SDHD has a nucleic acid sequence that encodes an amino acid sequence comprising a Gly12Ser mutation, a His50Arg mutation, a His145Asn mutation or a combination thereof.

The methods can further comprise obtaining a sample from the individual. A suitable sample for use in the methods of the invention is any sample obtained from the individual that comprises the individual's SDHB or SDHD. Examples of suitable samples include a tissue sample (e.g., organ, placenta), a cell sample (e.g., peripheral leukocytes; cell lysate), a fluid sample (e.g., blood, amniotic fluid, cerebrospinal fluid, urine, lymph) and any combination thereof. Methods of obtaining such samples and/or extracting SDHB and/or SDHD nucleic acid or protein from such samples are described herein and known to those of skill in the art.

As will be apparent to one of skill in the art, a variety of methods can be used to determine the presence of a mutated succinate dehydrogenase B (SDHB), a mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual or in a sample obtained from the individual.

In one aspect, the sequence of the SDHB, SDHD or combination thereof, in the individual is determined. A variety of methods for determining the nucleic acid and/or amino acid sequence of SDHB and/or SDHD can be used. For example, the amino acid sequence of the SDHB, SDHD or combination thereof is determined using polymerase chain reaction (PCR) amplification and direct sequencing (McWhinney, S. R., et al., J. Clin. Endocrinol. Metab., 89:5694-5699 (2004); Mutter, G. L., et al., J. Natl. Cancer Inst., 92:924-930 (2000)).

In addition or alternatively, the function of the SDHB, SDHD or combination thereof, in the individual is determined. There are also a variety of methods that can be used to determine the function of SDHB and/or SDHD. For example, in one aspect, the function of the SDHB, SDHD or combination thereof is determined by measuring the production of reactive oxygen species (ROS) wherein an increase in the production of ROS indicates the presence of a mutated SDHB, SDHD or a combination thereof. The increase in ROS production is at least about a 1-fold increase compared to a control. In another aspect, the increase in ROS production is at least about a 1.5-fold increase compared to a control. The production of ROS can be measured using, for example, using carboxy-H2DCFDA and confocal microscopy.

In another aspect, the function of SDHB, SDHD or a combination thereof is determined by detecting activation of an antiapoptotic/proproliferative AKT (protein kinase B) pathway, a mitogen-activated kinase (MAPK) pathway or a combination thereof. Activation of these pathways can be determined by detecting the presence of phosphorylated AKT, phosphorylated MAPK or a combination thereof, using, for example, one or more antibodies having binding specificity for the phosphorylated AKT or the phosphorylated MAPK.

The methods of the invention can further comprise comparing the presence of a mutated SDHB, SDHD or combination thereof in the individual to a control. Suitable controls for use in the methods provided herein are apparent to those of skill in the art. For example, a suitable control can be established by assaying the SDHB and/or SDHD sequence and/or function of one or more (e.g., a large sample of) individuals which do not have CS or CS-like syndrome. Alternatively, a control can be obtained using a statistical model to obtain a control value (standard value; known standard). See, for example, models described in Knapp, R. G. and Miller M. C. (1992) Clinical Epidemiology and Biostatistics, William and Wilkins, Harual Publishing Co. Malvern, Pa., which is incorporated herein by reference.

As used herein the term “individual” includes animals such as mammals, as well as other animals, vertebrate and invertebrate (e.g., birds, fish, reptiles, insects (e.g., Drosophila species), mollusks (e.g., Aplysia). Preferably, the animal is a mammal. The terms “mammal” and “mammalian”, as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses).

In one aspect of the invention, the individual is negative for a mutated phosphatase and tensin homolog deleted on chromsome ten (PTEN). In another aspect of the invention, the individual has elevated expression levels of maganese superoxide dismutase. In yet another aspect of the invention, the individual has a carcinoma of the breast, thyroid (e.g., papillary thyroid carcinoma), kidney (e.g., renal cell carcinoma) or a combination thereof.

The methods of detection of CS and/or CS-like syndrome can be used in combination with other methods used to detect CS and/or CS-like syndrome (e.g., operational diagnostic criteria of the International Cowden Consortium, Table 1).

The invention is also directed to an article of manufacture for detecting Cowden syndrome (CS) or Cowden-like syndrome in an individual, comprising one or more agents that detects mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, and instructions for use. In one aspect, the agent detects the sequence of the SDHB, SDHD or combination thereof. In another aspect, the agent detects the production of reactive oxygen species (ROS). In yet another aspect, the agent detects phosphorylated AKT, phosphorylated MAPK or a combination thereof using one or more antibodies having binding specificity for the phosphorylated AKT, the phosphorylated MAPK or a combination thereof.

EXEMPLIFICATION Materials And Methods Patients

Peripheral blood samples accrued from 375 CS and CS-like individuals, who were germline PTEN mutation negative after comprehensive mutation analysis which includes all 9 coding exons, flanking intronic regions, and minimal promoter region of PTEN and examination for large deletions and rearrangements, were utilized (FIG. 1). Classic CS was diagnosed when the operational diagnostic criteria of the International Cowden Consortium were met (Table 1) (Liaw, D., et al., Nat. Genet., 16:64-67 (1997)). The diagnosis of CS-like was made when an individual did not meet any of the strict diagnostic criteria but had features that were one or two criteria short of the operational diagnostic criteria (Table 1). Peripheral blood samples from 700 normal white populational controls of northern and western European origin, which were anonymized prior to storage and analysis were utilized. Informed consent was obtained for all subjects (CS/CS-like individuals and controls) in accordance with procedures and protocols approved by the respective Human Subjects Protection Committee of each participating institution. All subjects, whether CS/CS-like, or controls, participated on a voluntary basis. See Ni, Y., et al., Am. J. Hum. Genet., (AJHG), 83:261-268 (2008) which is incorporated herein by reference.

Mutation Analysis

Genomic DNA was extracted from peripheral leukocytes and PCR amplification and direct sequencing (ABI3730xI) of PTEN, SDHB, SDHC and SDHD were performed as previously reported (McWhinney, S. R., et al., J Clin. Endocrinol. Metab., 89:5694-5699 (2004); Mutter, G. L., et al., J. Natl. Cancer Inst., 92:924-930 (2000), which are herein incorporated by reference). It is important to note that all 700 controls had the entire sequence of SDHB, SDHC and SDHD sequenced and no variants identified.

Cell Lines and Cell Culture

Human immortalized lymphoblast cell lines obtained from patients and controls were cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS). All cell lines were cultured at 37° C. with 5% CO₂.

Protein Analysis

Whole-cell lystates were prepared using Mammalian Protein Extraction Reagent (Pierce, Rockford., Ill.) supplemented with protease inhibitor cocktail (Sigma). Lysates were either separated by SDS-PAGE and transferred to nitrocellulose or applied to nitrocellulose using a dotblot apparatus (BioRad). The resulting blots were then subjected to western blot analysis (Weng, L. P., et al., Hum. Mol. Genet., 11:1687-1696 (2002)) for either: SDHB (AbCarn, USA), MnSOD (Upstate Biotechnology, Waltham, Mass.), PTEN (Weng, L. P., et al., Hum. Mol. Genet., 11:1687-1696 (2002)) (Cascade Biosciences, Portland, Oreg., USA) P-MAPK, MAPK, AKT, P-AKT or actin (Cell Signaling Co, Beverly, Mass., USA). For the phosphorylation of MAPK an antibody that recognizes the activation phosphorylation of residues Thr187 and Thr189 of the p44-MAPK and the equivalent phosphorylation in p42-MAPK was utilized. For Akt phosphorylation, an antibody that recognizes the activation phosphorylation of Ser473 was utilized. Both of these antibodies are traditionally utilized to monitor phosphorylation, and thus activation, of these enzymes. Proteins were detected using ECL substrate (Amersham Biosciences., Inc., Chicago, Ill., USA) and autoradiography.

Confocal Microscopy

Images were collected with a Leica TCS SP2 AOBS confocal microscope (Leica Micro-Systems, Heidelberg, GmbH) using a HCX Plan Apo 63x/1.4NA oil immersion lens. The cells were excited with 488 nm light from an Argon laser and emitted light was collected between 500-550 nm. Collection parameters remained constant for all samples. Quantitation of ROS was performed by standard FACS (HFE), with controls normalized to 1.

Statistical Analysis

The frequency of each of the established CS-specific component carcinomas (breast and epithelial thyroid) and 2 of the strongly suspected component carcinomas (renal cell and endometrial) in SDHx mutation positive individuals were compared to that in a cohort of 230 PTEN mutation positive individuals with CS/CS-like phenotypes. Both groups were ascertained by identical clinical criteria as noted in the first section of the Methods. Fisher's 2-tailed Exact Test was applied with significance at p<0.05.

Results

To address the hypothesis, protein lysates from 375 PTEN mutation negative CS/CS-like individuals were screened for increased expression of manganese superoxide dismutase (MnSOD) because the latter is a good indicator and first screen for general (complex I-VI, especially complex II or V) mitochondrial dysfunction (FIG. 1). Dot blot analysis of these patients' protein lysates and 18 population controls identified 74 (20%) PTEN mutation negative patients with elevated MnSOD protein levels (FIGS. 1 and 2A). These 74 were subjected to SDHx mutation analysis (FIG. 1). Those that did not have elevated MnSOD levels were not included because a pilot study of 40 CS/CS-like PTEN mutation negative levels without elevated MnSOD were shown not to harbor any SDHx mutations (Eng et al., unpublished data). Of the 74 with germline elevations of MnSOD, 10 (13.5%; 95% confidence intervals(CI) 7.3-23.3%) were found to have germline mutations/variants in SDHB (N=3) or SDHD (N=7): Ala3Gly and Ser163Pro (N=2) in SDHB and His145Asn (N=1), His50Arg (N=2) and Gly12Ser (N=4) in SDHD (FIG. 2, Table 2). None of these SDH mutations were found in 700 normal controls (P<0.001, Fisher's 2-tailed exact test). All 3 genes, SDHB/C/D, were sequenced in the controls and no variants uncovered.

Then the 5 different SDH mutations/variants, identified in the 10 CS/CS-like individuals, were subjected to functional analysis (FIGS. 2A-2E and Table 3). First, the increased MnSOD protein levels noted on dot blot were confirmed by Western blot (Table 3). Because it is known that SDH dysfunction can result in increased production of reactive oxygen species (ROS) (Ishii, T., et al., Cancer Res., 65:203-209 (2005); Slane, B. G., et al., Cancer res., 66:7615-7620 (2006)), the pathogenicity of these different SDH mutations was also tested by direct measurements of increased ROS levels using (5-(and-6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and confocal microscopy (FIG. 2C, Table 3). SDHB Ser163Pro, SDHD Gly12Ser and His50Arg resulted in increased ROS levels (Table 3). SDHB Ala3Gly and SDHD His145Asn, in contrast, had normal ROS levels (Table 3).

PTEN is a tumor suppressor which down-regulates the anti-apoptotic/pro-proliferative AKT (protein kinase B) (Stambolic, V., et al., Cell, 95:29-39 (1998)) and mitogen activated kinase (MAPK) pathways (Weng, L., et al., Hum. Mol. Genet., 11:1687-1696 (2002)). Therefore, PTEN dysfunction is associated with activation of these pathways, whose downstream readouts include phosphorylated AKT (p-Akt) and MAPK (p-MAPK) (p42/44ERKs) (FIG. 2D). All 10 CS/CS-like patients with the germline SDHB/D mutations showed activation of AKT and MAPK manifested by increased phosphorylated AKT and MAPK in their germline, when compared to normal controls (FIG. 2D). Interestingly, the SDHD His145Asn mutation and the SDHB Ala3Gly mutation, which did not affect ROS, showed activation of the MAPK pathway and no or only mild activation of the AKT pathway (Table 3, FIG. 2E).

Renal cell carcinoma was present in 2/10 (20%; 95% CI 5-52%) CS/CS-like individuals with germline SDH mutations/variants (Table 2) compared to 4/230 (1.2%; 95% CI 0.5-4.5%) with germline PTEN mutations (p=0.03, Fisher's 2-tailed exact test). Epithelial thyroid carcinoma was found in 5 of 10 (50%; 95% CI 25-76%) SDH mutation positive individuals (Table 2) compared to 15/206 (7.2%; 95% CI 4-12%) with germline PTEN mutations (p<0.001). Interestingly, the histology of all the SDH-related thyroid cancers was papillary thyroid carcinoma compared to only one of the 15 thyroid cancers in PTEN mutation carriers (P<0.001). Female breast cancer was found in 6 of 9 (66.7%; 95% CI 36-88%) SDHx mutation positive women (Table 2) compared to 28% (95% CI 22-34%) of women with germline PTEN mutations (p<0.001). It is important to note that the frequencies of uterine endometrial carcinomas and uterine leiomyomas in our women with SDHx mutations were similar to those in women with PTEN mutations (P>0.05). One individual, the 55-year old man with germline SDHD His50Arg was incidentally found to have a unilateral pheochromocytoma.

Discussion

The observations described herein indicate that a subset of CS or CS-like individuals, without germline PTEN mutations, may be accounted for by germline mutations or variants in either the SDHB SDHD, but not SDHC, genes. SDHB and SDHD are the susceptibility genes for familial pheochromocytoma-PGL syndrome (Eng, C., et al., Nature Rev. Cancer, 3:193-202 (2003)). At least one of the 5 different mutations found in the 10 CS/CS-like individuals, SDHD His145Asn, has never been described before in individuals and families with pheochromocytoma and/or PGL (SDHx Mutation Database). Because these mutations are neither in dbSNP nor in our 700 control individuals, this almost certainly is a pathogenic germline mutation. His145 is also highly conserved through mouse, sheep and cow, arguing for the biological importance of this amino acid residue. Functional analyses corroborate the pathogenicity of this missense mutation. This mutation shows activation of the MAPK, but not AKT, pathway (Table 3, FIGS. 2D and 2E), mimicking PTEN dysfunction via the latter's nuclear role and/or protein phosphatase activity (Chung, J. H., et al., Cancer Res., 65:8096-8100 (2005)). Thus, taken together, these genetic and functional data represent strong evidence that SDHD His145Asn mutation lends susceptibility to PTEN mutation negative CS/CS-like disorders.

The SDHB Ala3Gly variant is shown in dbSNP from the Human Genome sequencing project, but no frequency is noted. The latter usually means it is an extremely rare variant or it may be found in a non-white population. Nonetheless, our germline Ala3Gly variant occurred in a CS/CS-like individual who is white of Northern/Western European ancestry and this variant is absent in 700 normal chromosomes originating from 700 white controls of the same ancestral background. More importantly, Ala3Gly results in obvious activation of the MAPK pathway and mild activation of the AKT pathway (FIG. 2E, Table 2). It would be important to note that dot blot analysis of >700 normal controls showed that none of these control samples had increased MnSOD, increased P-MAPK or increased P-AKT (Waite, Sadler and Eng, unpublished data). Thus, the functional data strongly suggest that Ala3Gly is pathogenic and may function in a low penetrance fashion in the CS/CS-like setting.

There are human genetic reports that both support and refute SDHD Gly12Ser and His50Arg, as pathogenic (Kytola, S., et al., Genes Chromosomes Cancer, 34:325-332 (2002); Perren, A., et al., Oncogene, 21:7605-7608 (2002); Cascon, A., et al., Genes Chromosomes Cancer, 37:220-221 (2003)).These 2 variants have been reported to occur in 1.1-3% of Spanish population controls. His50Arg has been described in 2-3% of a French Canadian cohort as well. SDHB Ser163Pro has been described in African Americans at a 2% frequency. However, none of our 700 control chromosomes, originating from whites of Northern and Western European ancestry, were found to harbor these 3 variants (p<0.001). None of our CS/CS-like patients nor any of our controls are of Spanish, French Canadian or African ancestry. More importantly, we have shown that these 3 variants do result in increased ROS levels (Table 3, FIG. 2). Of significance to this report, moreover, Ser163Pro, His50Arg and Gly12Ser all result in activated signaling down the AKT and MAPK pathways (FIG. 2E and Table 3), mimicking PTEN dysfunction although none of these samples had PTEN alterations. Our genetic and functional data, together with recent evidence showing that most rare missense variants are deleterious (Kryukov, G. V., et al., AJHG epub (2007)), therefore, indicate that these 3 variants are pathogenic at least in the context of our CS/CS-like individuals, and might either be a lower penetrance allele or also signal down other unknown pathways. While we have provided genetic and strong functional evidence to show that these 3 variants are pathogenic in the CS/CS-like context, how do we explain the 1.1-3% prevalence of Gly12Ser and His50Arg in the Spanish or French Canadian populational controls and the 2% prevalence of Ser163Pro in the black population? One hypothesis is that since these are populational controls (in contrast to healthy controls), these might be individuals who have CS/CS-like phenotypes and have not been recognized given that these syndromes are extremely difficult to diagnose and more plausibly, these are individuals with partial phenotypes (i.e., formes frustes), the latter of which are quite common in the general population.

DNA from family members is not currently available for testing segregation of the mutations with clinical phenotype within families, noting that 4 individuals do not have any family history. However, maternal imprinting of SDHD (Launonen, V., et al., proc. natl. Acad. Sci, USA, 98:3387-3392 (2001)) and decreased penetrance of SDHB (Launonen, V., et al., Proc. Natl. Acad. Sci, USA, 98:3387-3392 (2001)) mutations, even in classic familial pheochromocytopenia/paraganglioma syndromes, may make this type of family analysis, especially in this present setting, much less informative. Importantly, not only do these mutations likely cause some sort of mitochondrial dysfunction as evidenced by increased expression of MnSOD and/or increased ROS, but they also show increased signaling down the PI3K-AKT and/or MAPK pathways, the latter of which car occur with pathogenic PTEN mutations as well. The reason why not all individuals found to have increased MnSOD also have germline SDHx mutations is because MnSOD levels are a broad and general (and not necessarily specific) indication of mitochondrial complex I-VI (electron transport/respiratory chain) function.

Access to the tissues or tumors for the patients with germline SDH variants and mutations was not available. However, because of the continuing lack of understanding of SDH-related carcinogenesis, it may not be helpful to look for loss of the remaining wildtype allele in tumors of these current cases. In classic SDH-related pheochromocytopenia/paraganglioma syndromes, sometimes there is somatic loss of the remaining wildtype allele accompanying the germline mutation, but retention of the wildtype allele is also observed (Baysal, B. E., et al., Science, 287:848-851 (2000); McWhinney, S. R., et al., J. Clin. Endocrinol. Metab., 89:5694-5699 (2004)). Even more puzzling in the well-documented maternally imprinted SDHD-related tumors. With maternal imprinting, one would not expect to see loss of the remaining allele but mono-allelic expression of the mutant (paternally-transmitted) SDHD allele. In contrast, paraganglioma from germline SDHD mutation positive individuals still show clear bi-allelic SDHD expression (Baysal, B. E., et al., Science, 287:848-851 (2000)).

The precise mechanism leading to neoplastic transformation in patients with mutations of mitochondrial tumor suppressors is not fully understood. One hypothesis suggests that succinate, the substrate of SDH, functions as a second messenger between the mitochondria and cytosol, and inhibits the prolyl-hydroxylase enzymes, thus stabilizing HIF1 (Koivunen, P., et al., J. Biol. Chem., 282:4524-4532 (2007)). This inhibition could contribute to stabilization of HIF and promote transcription of genes containing hypoxic response elements (Selak, M. A., et al., Cancer Cell, 7:77-85 (2005)). An alternative hypothesis is mutations in SDH result in increased ROS (Ishii, T., et al., Cancer res., 65:203-209 (2005); Slane, B. G., et al., Cancer Res., 66:7615-7620 (2006)). This metabolic stress results in genomic instability and accumulation of HIF1 (Ishii, T., et al., Cancer res., 65:203-209 (2005)). Additionally, increased ROS levels can inactivate proteins, including PTEN, via protein oxidation (Lee, S. R., et al., J. Biol. Chem., 277:20336-20342 (2002)). Because of these reports and the finding described herein that germline SDHB/D mutations/variants in CS/CS-like individuals, it was hypothesized that at least a subset of germline PTEN mutations may also lead to mitochondrial dysfunction. As proof of principle, it was found that 5 of 11 (45%) patients with germline PTEN mutations actually had over-expression of MnSOD protein in the absence of SDH mutation (Patocs and Eng, unpublished data). While the observations and the existing literature indicate interesting signaling crosstalk between the PTEN and mitochondrial signaling pathways, it is also entirely possible that SDH-related CS/CS-like phenotypes might be unrelated to the PTEN-deficient mechanism and may represent a previously undescribed syndrome.

In summary, shown herein is that a subset of patients with CS or CS-like phenotypes likely has mitochondrial dysfunction irrespective of PTEN mutation status, and that this dysfunction can occur by different molecular mechanisms (FIG. 3). CS-associated SDHB or SDHD mutations may be associated with activation of similar anti-apoptotic pathways as observed with germline PTEN mutations, and that degree of mitochondrial dysfunction might differentially affect the AKT and MAPK pathways. Thus, failure of apoptosis regulation in patients, mediated by either germline PTEN or SDH mutations, resulting in mitochondrial dysfunction could be a unifying explanation for tumorigenesis in these patients (FIG. 3). Germline SDH mutation carriers have significantly higher frequencies of breast, thyroid and renal cell carcinomas compared to those with germline PTEN mutations (Table 1). It would be important to note that germline SDH mutation carriers have significantly higher frequencies of breast, thyroid and renal cell carcinomas compared to those without germline PTEN and without SDH mutations as well (Eng, unpublished data). In this study, all SDH-related thyroid cancers are papillary in contrast to PTEN-related epithelial thyroid cancers where all but one are follicular histologies. The frequencies of benign and malignant uterine disease were virtually identical between those with germline PTEN mutations and those with SDH mutations.

This data have important implications for both patient care and genetic counseling. Since 1997, the only susceptibility gene for CS and individuals with some neoplasias mimicking CS (CS-like) has been PTEN. Now, SDH is shown herein to be a susceptibility gene for a subset of PTEN mutation negative patients with tumors reminiscent of those component to CS. Because this study has only analyzed in detail 10 SDH mutation positive individuals with CS/CS-like features, these data should be further validated. Until then however, it appears that mutation positive CS/CS-like patients and their families have significantly increased risks of carcinomas of the breast, thyroid (especially papillary thyroid carcinoma) and kidney beyond those of PTEN-related CS. Germline PTEN mutation-negative CS/CS-like individuals should be offered SDH testing, especially in the setting of breast, papillary thyroid and/or renal carcinomas (FIG. 4). Clinicians should consider annual renal ultrasounds and PGL-type surveillance, beyond the NCCN practice guidelines for PTEN hamartoma tumor syndrome, should an individual with tumors similar to those found in CS carry a germline SDHx mutation or variant (FIG. 4).

TABLE 1 International Cowden Syndrome Consortium Operational Criteria for the Diagnosis of Cowden Syndrome (Ver. 2006) Pathognomonic Criteria Major Criteria Minor Criteria Mucocutaneous lesions: Breast carcinoma Other anatomic thyroid Trichilemmomas, facial Epithelial thyroid lesions Acral keratoses carcinoma Mental retardation Papillomatous papules Endometrial carcinoma (say, IQ ≦75) Mucosal lesions Macrocephaly (say, GI hamartomas Lhermitte-Duclos disease ≧97% ile) Fibrocystic disease of the breast Lipomas Fibromas GU tumors or malformation Operational Diagnosis in an Individual 1. Mucocutaneous lesions alone if: a) there are 6 or more facial papules, of which 3 or more must be trichilemmoma, or b) cutaneous facial papules and oral mucosal papillomatosis, or c) oral mucosal papillomatosis and acral keratoses, or d) palmo plantar keratoses, 6 or more 2. 2 Major criteria but one must include macroencephaly or LDD 3. 1 Major and 3 minor criteria 4. 4 Minor criteria

TABLE 2 Genotype and clinical phenotype for PTEN mutation negative patients with germline SDHB or SDHD mutations/variants SDH Genotype Patient's Clinical Features Age/Sex Gene Mutation Breast Thyroid Renal Uterus Family History 41F SDHB Ala3Gly C L Endometrial cancer 29F SDHB Ser163Pro C B Breast Cancer, PTC 54F SDHB Ser163Pro C B Breast Cancer, PTC 69F SDHD Gly12Ser C C Breast Cancer, Endometrial 62F SDHD Gly12Ser B B C L None 46F SDHD Gly12Ser C C L None 42F SDHD Gly12Ser C L None 56F SDHD His50Arg C C Breasat Cancer 55M SDHD His50Arg C Breast Cancer, PTC 53F SDHD His145Asn C B C None C carcinoma, B benign patholo^(g)y, L uterine leiornyomas. PTC family history of papillary thyroid carcinoma

TABLE 3 Identified Gerniline SDHBor SDHD mutations/variants in PTEN mutation negative CS/CS-like individuals and their functional consequences P-Akt Fold P-MAPK Fold Mutation/Variant MnSOD* ROS Change Change SDHB Ala3Gly Increased Normal 1.2 1.3 SDHB Ser163Pro Increased Increased 2.7 1.7 SDHD Gly12Ser Increased Increased 1.9 1.9 SDHD His50Arg Increased Increased 2.0 1.7 SDHD His145Asn Increased Normal 1.0 1.2 *Note patients chosen for SDH analysis were selected for increased MnSOD protein expression.

Fold change values represent the mean of normalized densitometrically obtained expressional levels of patient sample(s) relative to controls (where P-Akt/actin or P-MAPK/actin is set to 1.0), ie, a ratio of ratios.

ROS measurements were quantitated and normalized against controls (latter set at 1). The 3 mutations resulting in increased ROS had 1.5-fold over controls.

See legend to FIG. 2 for further details.

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. An article of manufacture for detecting Cowden syndrome (CS) or Cowden-like syndrome in an individual, comprising one or more agents that detects mutated succinate dehydrogenase B (SDHB), mutated succinate dehydrogenase D (SDHD) or combination thereof in the individual, and instructions for use.
 2. The article of manufacture of claim 1 wherein the mutated SDHB is an Ala3Gly mutation, a Ser163Pro mutation or a combination thereof.
 3. The article of manufacture of claim 1 wherein the mutated SDHD is a Gly12Ser mutation, a His50Arg mutation, a His145Asn mutation or a combination thereof. 